What can SNP-based heritability tell us about the etiology of AD today?

Here’s a nice article from Baker et al 2022, “What does heritability of Alzheimer’s disease represent?”

Here is a summary of some of their findings:

– The heritability of Alzheimer’s disease is complex and variable, depending on factors like age and population. Regarding age, in samples with older participants, there is a greater likelihood that AD genetic factors will play a role in disease risk.

– SNP-based heritability estimates decrease by 12% when APOE is excluded. When all other genome-wide significant hits were removed, SNP-based heritability only decreased by 1%.

– In APOE e44 carriers, the average age of onset is about 68 years, whereas for APOE e4 non-carriers, it is about 84 years. The authors suggest that for the APOE e4 non-carriers, disease burden is mostly due to the aggregate effect of a large number of common SNPs as well as comorbid disorders.

– When they restricted SNPs to a microglia gene set that was only 3% of SNPs, it still explained between 50% and 93% of the SNP-based heritability. This strongly suggests that microglia are critical for the mechanism of AD. It would have been nice to see this compared to gene sets for other cell types, such as neurons or oligodendrocytes, which could have given a better sense of how variability in the function of these different cell types may contribute to AD risk and also helped to assess the robustness of their methods.

– The authors suggest that for neurodegenerative disorders, heritability estimates should be adjusted for the age-related prevalence of cases. This would help to account for the genetic liability for the disease of individuals who do not yet show symptoms.

– The article provides helpful insights into the complex nature of Alzheimer’s disease heritability. It highlights the need for further research to identify biologically relevant AD gene-sets/pathways that could increase the signal-to-noise ratio by highlighting the most influential SNPs/genes in AD.

Most of the risk factors associated with Alzheimer’s disease from observational studies are likely wrong

Interesting article from Korologou-Linden et al 2022, “The causes and consequences of Alzheimer’s disease: phenome-wide evidence from Mendelian randomization”.

In this study, the authors used a Mendelian randomization approach to examine the causal relationships between various risk factors and Alzheimer’s disease. They used the UK Biobank, which has a massive sample size of >300,000 participants.

They found that genetic variation at one gene — APOE — is far and away the main mediator of Alzheimer’s disease genetic risk. This replicates why Alzheimer’s has been called a quasi-monogenic disease — APOE has that large of an effect.

They don’t hold anything back in the discussion, basically arguing that their study disagrees with observational studies because their methodology is better and observational studies are wrong, because observational studies can’t identify causality.

Instead, they suggest that associations with Alzheimer’s disease in observational studies are due to reverse causation (i.e. they are symptoms of early/prodromal Alzheimer’s disease, rather than causes) or simply due to selection bias.

This means that the things often associated with Alzheimer’s disease in observational studies — body mass index, blood pressure, and physical activity — might not actually increase the risk of disease. Interestingly, Alzheimer’s genetic risk in this study is actually associated with a lower body mass index and body fat in people 53-72 years old.

They also found that Alzheimer’s disease risk is associated with a lower fluid intelligence score, with no causal effect on educational attainment.

One caveat I have with the study is that I’d like to learn about the associations of these risk factors with other forms of cognitive impairment. Laypeople often use the term “Alzheimer’s” to refer to dementia or age-related cognitive impairment in general.

For example, does a higher genetic risk for elevated blood pressure or body mass index causally affect the risk for vascular-associated cognitive impairment? My guess is that they might, which might cause this study to be a bit misleading if the results were taken in the wrong way.

That said, there seems to be a real effect of APOE on the risk of cognitive impairment that is independent of classical risk factors body mass index, blood pressure, and physical activity. And this study helps to parse out how that might be occurring, which will hopefully help to develop better preventive approaches and treatments.

Extracellular space preservation with immersion fixation of brain sections

Here’s a nice article from 2021: “Permeabilization-free en bloc immunohistochemistry for correlative microscopy” by Fulton and Briggman. A few thoughts:

1. The protocol helps to overcome an impediment to correlative en bloc EM and IHC by enabling the ultrastructural preservation of brain tissue and antibody penetration into relatively thick tissue sections, without the use of permeabilizing agents. This could help to facilitate the use of pre-embedding IHC in ultrastructural analysis techniques such as 3D EM.

They replicate the finding that permeabilization dramatically decreases ultrastructural quality, so should be avoided if possible:

https://elifesciences.org/articles/63392, Figure 1

They argue that a key way they were able to accomplish antibody penetration without permeabilization was via the preservation of the extracellular space (ECS).

2. The brains sections they used are still quite thin relative to those that are practical in neuropathology, at 300 um – 1 mm. In neuropathology, human brains are somewhat frequently sectioned at 5 mm, but even that is challenging and requires expert skill.

One could try to use a device such as a compresstome to help with the sectioning process. Here’s a video showing how the compresstome works on mouse brains. But it seems difficult to scale this to human brain sizes.

(If one were to achieve such thin sections in a high-fidelity way, you could theoretically cryopreserve them with vitrification or near-vitrification procedures and therefore avoid fixation altogether. Although, avoiding fixatives would also make room temperature preservation not currently possible.)

3. Another possible mechanism for why their protocol worked, that the authors did not discuss as far as I could tell, is that tissue decomposition during the immersion fixation process — which is slower than perfusion fixation — may itself cause membrane permeabilization. With a long enough time period of decomposition, cell membrane breakdown is an inevitable event, so the question is really whether the immersion fixation was slow enough to allow it to occur. My guess is that it was a contributing factor.

This may also help to explain why some epitopes are more accessible (eg Homer) than others (eg PSD-95). If a protein is a part of stronger gel-like networks, this gel-like network will likely break down slower during the decomposition process, and therefore be more difficult for antibodies to access without permeabilization.

4. Do we even need immersion fixation for ECS preservation? They cite Cragg 1980 as an example of a study that achieved ECS preservation using perfusion. It’s still not entirely clear to me why perfusion doesn’t usually achieve ECS preservation, but it seems like it probably depends on the osmotic concentration of the perfusate. Cragg 1980 is 30+ years old now; it would be ideal if it could be replicated and the phenomenon understood better.

Prenatal epigenetic age acceleration in Down syndrome

That’s a result of Xu et al 2022, “Accelerated epigenetic aging in newborns with Down syndrome”.

This study furthers our understanding of a syndrome of accelerated aging. The authors show a significant acceleration of an epigenetic aging marker in the blood of people with Down syndrome. Furthermore, they show that this effect is present at birth and is significantly stronger in newborns who have Down syndrome plus GATA1 mutations. This association with GATA1 mutations is intriguing as GATA1 mutations are associated with transient abnormal myelopoiesis. One thing that this study does not do is investigate the mechanism by which this age acceleration occurs.

One hypothesis based on this finding is that it might help explain why people with Down syndrome have an increased susceptibility to Alzheimer’s disease. Lore has long been that this is due to the triplication of amyloid precursor protein, however, this study suggests that age acceleration may also play at least a part in the increased susceptibility of people with Down syndrome to aging-associated cognitive impairment and Alzheimer-type neuropathology.

from https://onlinelibrary.wiley.com/doi/10.1111/acel.13652

Correlating immunohistochemistry with serial block-face electron microscopy of neurons

In Talapka et al 2022, “Application of the mirror technique for block-face scanning electron microscopy”, the authors use a modified “mirror” technique to combine immunohistochemistry for labeling of dendrites and ultrastructural analysis in 3D-EM of osmicated sections. This relies on the finding that the surface of a tissue block can still be imaged using confocal microscopy. The authors show that the cell body of a somatostatin immunopositive neuron and one of the emerging dendrites can be clearly visualized and reconstructed after the use of their technique. It is likely that the dendritic arbor of a large number of neurons can be analyzed using this technique. The technique combines the advantages of a high-resolution approach and of a labeling method for specific cellular markers. The morphological preservation of the structures seen on the surfaces of tissue sections such as blood vessels will in part determine the quality of the images. Here is one of the figures from their paper:

image from https://link.springer.com/article/10.1007/s00429-022-02506-w

Integrating synchrotron microtomography with electron microscopy in the study of mammalian brain tissue

Bosch et al 2022, “Functional and multiscale 3D structural investigation of brain tissue through correlative in vivo physiology, synchrotron microtomography and volume electron microscopy”, is an interesting study that brings X-ray microscopy to bear on the problem of correlating structure and function.

The authors studied hippocampal CA1 and olfactory bulb circuits via multiple imaging modalities, including 2-photon calcium imaging, X-ray microscopy, and serial block-face electron microscopy. In all cases, the imaging modalities had different strengths in identifying different circuit elements, and the authors were able to correlate structure and function in interesting ways. The interplay between structural, functional, and molecular-level data will be increasingly critical in systems neuroscience, and this study highlights some important points.

The authors should be commended on showing that X-ray microscopy can be used without causing significant damage on fixed and osmium/uranium/lead en-bloc EM embedded tissue, which is an important advance. The authors also showed that X-ray microscopy can be used at high resolution on thick mammalian brain tissues; this is important because X-ray microscopy has the potential to provide structural details at the level of individual dendrites, which is possible with volume electron microscopy but less easily scalable. Finally, the authors point out that staining protein and lipid distributions defines the ultrastructure of the tissue; this is an important point that is often missed.

Figure 4 from Bosch et al; https://www.nature.com/articles/s41467-022-30199-6

Question #21: How far would a typical molecule diffuse in a millisecond?

What is diffusion?

Diffusion is a type of passive transport that involves the net movement of molecules or ions from an area of higher concentration to an area of lower concentration down a concentration gradient. The concentration gradient is the difference in concentration between two points.

In biology, diffusion plays an important role in many biological events such as molecular transport, cell signaling, and neurotransmitter movement across a synaptic cleft.

How far would a typical molecule diffuse in a millisecond? A second? An hour?

Diffusion is a description of how molecules will randomly move around in a liquid. Their movement will be limited if they hit a barrier or randomly collide with another molecule and react, which is not described by diffusion.

The distance a molecule will diffuse in a certain amount of time depends on the size of the molecule, the viscosity of the fluid, and the temperature.

This can be explained by the Stokes-Einstein relation: D = kT/(6πηa), where:

– D = the diffusion constant

– k = the Boltzmann constant

– T = the temperature

– η = the viscosity coefficient of the fluid

– a = the radius of the diffusing molecule

The constant value is 6 assuming that the radius of the diffusing molecule is greater than the radius of the solvent.

Assuming that we are talking about diffusion at 25° C and in water, then there is a nice calculator on physiologyweb.com that lists diffusion coefficients for different ions and molecules:

https://www.physiologyweb.com/calculators/diffusion_time_calculator.html

If we are talking about the diffusion of a small molecule neurotransmitter such as glutamate, it has a MW of 147, which is close to glucose’s MW of 180. So we can use glucose’s diffusion coefficient as a rough guide for the diffusion of some types of small molecule neurotransmitters.

This calculator suggests that glucose will diffuse 1000 nm in a millisecond, 31,000 nm (31 μm) in a second, or 1,900,000 nm (1.9 mm) in an hour.

Molecular diffusion rates are helpful when building intuition about what structural information is necessary to be able to infer in brain preservation. Because, in the way that I think about it, molecular events that occur more slowly than rapid long-term memory recall can be instantiated (which, conservatively, can occur in ~500-1000 ms) cannot be uniquely necessary for the structural information describing it.

Inspired by CalTech’s Question #21 for cognitive scientists: “What is diffusion? How far would a typical molecule diffuse in a millisecond? A second? An hour? How does the diffusion equation differ from the cable equation?”

Question #20: What does the cable equation mean for neurons?

A simplified explanation of capacitance in neuronal membranes is that higher capacitance will tend to will cause a flow of ions towards the membrane on the cytoplasmic side due to the difference in charge across the membrane, called a displacement current; https://en.wikipedia.org/wiki/Cable_theory#/media/File:NeuronCapacitanceRev.jpg

– Cable theory can be derived in part from Ohm’s law, the fundamental theory of electricity that models the current flowing between two points as equal to the voltage distance between the two points divided by the material’s resistance, or in other words, the classic equation V = IR.

– The greater the cross-sectional area of the neurite’s cytosol (the interior part of it, containing biomolecules, electrolytes, and other ions), the easier an ion can flow through it, so the neurite’s longitudinal resistance, r_l, will be lower.

– If the cell membrane is more resistant to ion flow into or out of the cell (due to high membrane resistance, r_m), then charge will tend to accumulate inside the cell, and it will have a higher ionic current flowing down longer distances in the neurite. This is often represented by a paremeter called the length current, λ, equal to the square root of r_m divided by r_l.

– If a cell membrane has a lower membrane capacitance (c_m, which is usually a fairly constant value), then the relative ion flow down the neurite will be greater, due to a lower displacement current. How quickly the membrane voltage changes in response to a current injected at at given point can be predicted by the time constant, τ, equal to the product of c_m times r_m.

– An electrotonic potential results from a local change in ion conductance, e.g. after a synaptic event, that does not propagate. It becomes exponentially smaller as it spreads. This is opposed to an action potential, which reaches the voltage threshold by which it does propagate down the neurite (due to the opening of voltage-gated ion channels), and then spreads like a wave.

– Dendritic trees can perform non-linear integration of signals that can be predicted on the basis of cable theory. The existence of subthreshold membrane potential fluctuations in dendrites, which based on my understanding should dominate neuronal signaling, can allow variations in synaptic weight distributions and input timing to encode a substantial amount of information within a single neuron.

Inspired by CalTech’s Question #20 for cognitive scientists: “Derive the cable equation (for a uniform cylinder, with optimal boundary conditions). What does it mean for neurons?”

Question #19: Ion channel biophysics

What are the biophysics of voltage-gated sodium channels? 

Sodium channels are a major component of excitable membranes. They are an intrinsic component of excitable tissue that allows them to generate and propagate action potentials. These electrical signals are essential for proper neuronal communication.

The channel looks like a barrel, with 4-fold symmetry, and a diameter of about 10 nm. The channel has an activation gate, through which sodium ions can flow through. If the activation gate is closed, no ions can pass through, but if it is open, ions can pass through the pore. The channel is closed at rest, wherein the membrane voltage potential is polarized. When a sufficient voltage depolarization across the membrane occurs, the membrane will draw the gates open, allowing sodium ions to flow through and leading to further depolarization. When enough sodium has passed, the further voltage change causes the inactivation gate to close, thus stopping the flow of sodium ions and leading to repolarization.

Sodium channels are selective for sodium ions because the inner filter of the pore is highly negatively charged; the Na+ ion has a positive charge and will bind well to the inner filter. K+ ions, while also positively charged, cannot pass through because of a size restriction. The gate is not large enough for them to fit through. For ions to pass, they need to be smaller than the diameter of the filter; for ions with a larger diameter to pass, the filter would need to enlarge; however, the size of the filter cannot increase because the pore has a fixed size. These are the unique properties of the sodium channel that allow it to selectively conduct sodium.

Sodium channels are good targets for many drugs and toxins. For example, tetrodotoxin specifically binds to voltage-gated sodium channels and can stop sodium channels from opening, thereby blocking all neural signaling. 

What are the biophysics of transmitter-gated channels? 

Transmitter-gated channels are opened by transmitters. They are then generally ion-selective. To open the channel, the transmitter needs to bind to the receptor. The transmitter binding causes an allosteric change that allows another part of the channel to open, known as the ion channel gate. When open, the ion channel gate allows specific ions to pass through.

A special example is the NMDA receptor. Under normal circumstances, the NMDA receptor is blocked by Mg2+ and Zn2+ ions. When the post-synaptic neuron is depolarized, however, Mg2+ and Zn2+ ions are repelled. In this case, the receptor can be activated by glutamate. When activated, the NMDA receptor allows positive ions to pass through (K+, Na+, and Ca2+ ions), which can help sustain depolarization and lead to intracellular signaling events such as long-term potentiation.

NMDA receptors are often called “coincidence detectors” because these two events must occur together for the channel to open. First, the NMDA receptor must be activated by the post-synaptic being depolarized, and second, glutamate must be released.

Another example is the nicotinic acetylcholine receptor. When acetylcholine binds to the receptor, the channel opens. This allows sodium and potassium ions to pass through, which leads to depolarization and therefore a neural signal.

Most types of ion channel activity in the brain need regulation. Regulation can occur post-translationally through the addition of a phosphate group to one or more amino acids. The addition of a phosphate group to a particular location of the AMPA receptor, for example, has been shown to increase the probability of AMPA channel opening. The Ca2+/calmodulin kinase II pathway is able to phosphorylate the GluA1 AMPA receptor subunit at Ser831, causing an increase in AMPA channel conductance.

In addition to the post-translational regulation of channel activity, many channels are regulated by endogenous compounds in the brain. Serotonin is a monoamine neurotransmitter that regulates various types of sodium channels and potassium channels. Dopamine is also a monoamine neurotransmitter, and it can be found in extrasynaptic regions. Dopamine has been shown to increase potassium channel activity by activating dopamine D1 receptors in axons.  

Together, the biophysics of ion channels allow for neural signaling by allowing for the passage of ions into and out of the cell. This allows for changes in membrane potential and intracellular signaling. 

Inspired by CalTech’s Question #19 for cognitive scientists: “Describe the main biophysical characteristics of ionic channels. How does its biophysical properties contribute to its physiological function? What is thought to be the basis for the channels ion selectivity?”

Microwave irradiation as a brain banking tool

I love random reading old papers. They’re like treasure chests into secret knowledge that others might overlook. Here’s one: Guidotti et al 1974, “Focussed microwave radiation: a technique to minimize post mortem changes of cyclic nucleotides, dopa and choline and to preserve brain morphology”.

As a summary, they found that microwave irradiation in rat and mouse brains for 2 seconds led to the total inactivation of the enzymes involved in the regulation of numerous brain metabolites, such as cyclic AMP, cyclic GMP, choline acetyltransferase, and phosphodiesterase. As a result, it allowed for the accurate dissection of different brain nuclei for measurement of the concentrations of a variety of different metabolites in a variety of brain areas.

Rapid inactivation of enzyme activity; https://doi.org/10.1016/0028-3908(74)90061-6

The microwave irradiation method described by Guidotti et al. 1974 was relatively crude, as the authors only described microdissection of brain nuclei. It is unclear whether delicate cellular features such as synapses might also be preserved.

Interestingly, microwave irradiation produced extensive denaturation of proteins throughout the brain. Because since enzymes control metabolism, the inactivation of these enzymes can minimize changes in metabolism.

Microwave radiation could theoretically be combined with other brain preservation methods, such as fixation or cryopreservation, to minimize enzyme-driven autolysis during the procedure. A potential limitation of this method is that microwave heating is limited by the thermal conductivity of the tissue.